Project Summary The longterm goal of this project is to uncover how the niche for a stem population of vascular smooth muscle cells (VSMCs) is established during embryogenesis. It is becoming clear that a mesothelium origin of VSMCs is conserved by many vascular systems throughout the body, such as the heart, intestine, mesentery, lung, and liver. In the adult, the mesothelium generates VSM stem cells and VSMCs during wound healing. Despite the importance that the mesothelium in sourcing of VSMCs, little is known about how the mesothelium niche is induced and defined at the body cavity lumen and internal organ surface. We have recently developed a comprehensive high resolution live imaging system that enabled us for the first time to view body cavity formation within embryos. None of currently known mechanisms for lumen formation, such as fusion of intracellular vacuoles; ion channel-mediated osmolality changes; and apoptosiswere detected. Instead, we have found that: (a) The lateral plate mesoderm (LPM) and the endoderm produced long filamentous projections, that resembled specialized cytoneme filopodia (CF); (b) LPM's CFs extended through Laminin1-rich extracellular matrix (ECM), reached to the ectoderm, and directly received BMP7 from the ectoderm; (c) Endodermal CFs extended toward the ventral LPM; (d) Upon direct inter-germ layer contacts of the CFs, small openings appeared horizontally in the LPM. Their subsequent fusions established a single continuous body cavity lumen; (e) Factors ephrinA2 and ephrinB1 were expressed asymmetrically along the dorsoventral axis (the LPM was sandwiched by ephrinA2+/ephrinB1- ectoderm and ephrinA2-/ephrinB1+ endoderm); and finally (f) Of several ectodermal morphogens, ectodermal BMP7 was crucial for all key steps listed above for body cavity formation to initiate VSM stem cell niche development. Based on these surprising findings, this application proposes to experimentally test the following novel models for mesothelial niche development: The mesothelial niche housing VSM stem cells and VSMCs is initiated by two asymmetric signaling mechanisms along the dorsoventral axis: (1) CF-mediated inter-germ layer transfer of signaling factor(s) from the ectoderm and endoderm to the mesoderm; and (2) Morphogenetic separation of the LPM by an asymmetric expression of the Eph/ephrin gene family. The proposed study offers a new mechanistic model of asymmetric cellular and molecular interactions between the LPM and the overlaying ectoderm and underlying endoderm, and how this establishes the mesothelial niche that harbors VSM stem cells. The outcomes of this study will also serve as a new framework to design future therapeutic approaches for treatment of vascular diseases.